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EC number: 202-992-2
CAS number: 101-96-2
The objectives of this study were to determine the potential of
N,N’-Di-sec-butyl-p-phenylenediamine to induce developmental
toxicity after maternal exposure during the critical period of
organogenesis and to characterize maternal toxicity at the
exposure levels tested when given orally by gavage to time-mated
female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In
addition, the No Observed Adverse Effect Levels (NOAELs) for
maternal toxicity and developmental toxicity were evaluated.
The dose levels in this study were selected to be 0, 10, 30,
60 mg/kg/day, based on the results of the dose range finder (Test
Facility Study No. 20140512)
Chemical analyses of formulations were conducted once during
the study to assess accuracy and homogeneity.
The following parameters and end points were evaluated in
this study for the F0-generation: mortality/moribundity,
clinical signs, body weights, food consumption, gross necropsy
findings, organ weights (liver), number of corpora lutea, (gravid)
uterine weight and uterine contents.
In addition, the following parameters were determined for
the F1-generation: the number of live and dead fetuses,
early and late resorptions, total implantations, fetal body
weights, sex ratio, external, visceral and skeletal malformations
and developmental variations.
Formulation analyses confirmed that formulations of test item in
Polyethylene glycol 400 were prepared accurately and homogenously.
No mortality occurred during the study period.
Test item-related clinical signs were observed in all treated
Persistent green discoloration of the urine was observed among all
treatment groups starting at 10 mg/kg. This
clinical sign was noted in all treated animals at 30 and 60 mg/kg,
after 1-2 days of treatment (from Day 7-8 post-coitum onwards) and
in 15 out of 22 animals at 10 mg/kg, on average starting during
the second week of treatment. Although no clear
cause could be attributed to this finding, it was considered
not to be toxicologically relevant since clear signs of systemic
toxicity would have been observed in case of renal toxicity among
animals presenting with this abnormal colour in urine (no relevant
clinical signs were observed in any of the Group 2 and 3 animals
and in 18 out of 22 animals in Group 4 presenting with green
At 60 mg/kg, 4 out of 22 animals were noted with hunched posture
and/or piloerection for several consecutive days, with one of them
additionally presenting with reflux on one single day (Day 13
post-coitum), flat gait for 8 consecutive days, slight and
transient laboured respiration and rales for 1-3 days together
with slight pale ears and ptosis for 1-2 days.
At 60 mg/kg, mean body weight gain and food consumption were
statistically significantly lower than control mean during the
first days of treatment (over Days 6-9 post-coitum), followed by a
recovery afterwards. As complete recovery was noted, the
transient decrease in mean body weight gain and food consumption
was not considered adverse. From Day
12-15 post-coitum onwards a significant increase in food
consumption was observed at 60 mg/kg (15-16%vscontrol),
that might have occurred to compensate for the initial decrease in
food intake observed during the first days of the treatment period.
No toxicologically relevant changes in mean body weight, body
weight gain and food consumption were observed at 10 and 30 mg/kg
over the entire study period. The slight
increase in food consumption (<10%vscontrol) observed at
30 mg/kg over Days 12-21 post-coitum was considered non-adverse.
Macroscopic observations at necropsy did not reveal any
alterations that were considered to have arisen as a result of
treatment up to, and including, 60 mg/kg.
A test item-related increase inmean liver weight and
liver/body weight ratiowas observed in a dose-response
manner. Relative mean liver weights were 23% and 37%
higher than concurrent control mean at 30 and 60 mg/kg,
respectively. In a previously performed
Reproduction/Developmental Toxicity Screening Test by oral gavage
in Wistar Han rats with N,N’-Di-sec-butyl-p-phenylenediamine (Test
Facility Study no. 20140515) a test item-related increase in mean
relative liver weight was also observed in females treated at 30
and 60 mg/kg for at least 28 days (15% and 27%vscontrol,
respectively), together with incidental/background hepatic
microscopic findings at 30 mg/kg and non-adverse test item-related
microscopic alterations at 60 mg/kg (hepatocellular hypertrophy
with increased incidence and/or severity of cytoplasmic
rarefaction). However, thepossible adversity of
thedose-dependent moderate increase in liver weightobserved in the
current study was not assessed histopathologically and was,
therefore, not taken into account when determining the maternal
The number of pregnant females, corpora lutea, implantation sites,
and pre-implantation loss in the control and treatment groups was
in the range of normal biological variation.
There were 2 females, one each at 30 and 60 mg/kg that had an
early delivery on the day of scheduled necropsy (1 viable pup + 11
viable fetuses and 4 viable pups + 10 dead fetuses, respectively). Although
this number of fetal deaths was noticed in a female that delivered
early on the day of schedule necropsy (Day 21 post-coitum), a
relationship to treatment could not be completely discarded as it
occurred in the high dose group.
At 60 mg/kg, mean number of viable fetuses was statistically
significantly lower when compared to concurrent control mean (9.3vs11.4). The
% per litter of viable fetuses in this high dose group was also
lower when compared with concurrent control mean (89.1%vs97.5%)
and the 5thpercentile of the historical control data
(90.8%). This was considered to be related to
the higher incidence of early resorptions (caused by 2 litters;
each consisting of 9 and 12 early resorptions), resulting in a
higher litter incidence of post-implantation loss at 60 mg/kg. While
maternal toxicity may have played a role in the increase in early
resorptions in the two affected dams in the 60 mg/kg group, it was
not possible to assign a cause and effect relationship nor was it
possible to rule out a direct effect of the test item on the
survival of the embryo.
Mean combined fetal body weights were 8% and 6% highervscontrol
mean at 30 and 60 mg/kg, respectively, and values were outside the
historical control range. The increase in mean
fetal body weights in both sexes could partly be explained by the
slightly higher mean food consumption (absolute and relative)
noted in females at 30 and 60 mg/kg from Day 12-15 post-coitum
onwards. This observation in fetal body weight
was considered not to be of toxicologically relevance due to the
slight magnitude of the change (less than 10%), absence of test
item-related malformations or detrimental developmental variations
and as the opposite effect (i.e. a decrease) would be expected in
case of intrauterine/fetal growth retardation.
No statistically significant changes in fetal body weights (both
sexes) were noted at 10 mg/kg.
The treatment-related decrease in the skeletal variation
bent ribs observed at 30 and 60 mg/kg could be related by the
higherfetal body weightobserved in both groups. This
variation was considered not to be toxicologically relevant as
incidences were within the historical control range and since a
lower incidence for bent ribs is not considered to have any
No treatment-related changes were noted in any of the remaining
developmental parameters investigated in this study (i.e. sex
ratio, external and visceral malformations and developmental
variations, and skeletal malformations).
In conclusion, based on the results in this prenatal developmental
toxicity study the maternal No Observed Adverse Effect Level
(NOAEL) for N,N’-Di-sec-butyl-p-phenylenediamine was established
as being 30 mg/kg (based on clinical signs at 60 mg/kg).
The developmental NOAEL for N,N’-Di-sec-butyl-p-phenylenediamine
was established as being 30 mg/kg (due to the higher incidence of
early resorptions in 2 females at 60 mg/kg).
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